Although skeletal muscle insulin resistance is a primary contributor to the development of metabolic abnormalities and chronic diseases, such as type 2 diabetes and cardiovascular disease, the molecular mechanisms of its pathogenesis remain elusive. Defects in the phosphorylation and dephosphorylation events in the insulin signaling pathway, such as serine/threonine phosphorylation of Insulin Receptor Substrate-1 (IRS-1), are considered to be one of the main causes of insulin resistance. Most research on the regulation of these phosphorylation events has been focused on kinases and is performed in cell culture or animal models. We hypothesize that serine/threonine protein phosphatase 2A (PP2A) complexes in human skeletal muscle are over-activated and dysregulated in insulin resistance and type 2 diabetes. This hypothesis is formulated based on the existing literature suggesting hyperactivation of PP2A under the duress of glucolipotoxicity and diabetes in cell/animal models, and the following novel preliminary findings of our own: 1). The catalytic subunit of PP2A (PP2Ac) associates with IRS-1 in skeletal muscle of lean healthy participants, and this interaction is significantly increased in obese/overweight and T2D muscle; 2), Insulin suppresses PP2Ac activity and Leu309 carboxylmethylation in muscle of lean healthy participants, but not type 2 diabetic patients. To test our hypotheses, we will assess the activity (Aim 1), post-translational modifications (Aim 2), and interaction partners (Aim 3) of PP2A in skeletal muscle from lean healthy controls (LC), obese nondiabetic subjects (OC), and type 2 diabetic patients (T2D), using state-of-the-art HPLC-nanospray-tandem mass spectrometry (HPLC-ESI-MS/MS). The overall goal is to address the knowledge gap of phosphatase function and regulation in insulin resistance and T2D in humans, using a combination of clinical, biological, and proteomic approaches developed in our laboratory. For Aim 1, we will perform PP2Ac co- immunoprecipitation experiments followed by assay of PP2A activity in muscle biopsies from the 3 groups under basal and insulin infusion. For Aim 2, we will assess pTyr307 and mLeu309 in the 3 groups using Western blot analysis. In addition, we will employ the proteomics approach for phosphorylation quantification developed in our laboratory to provide a global view of PP2Ac phosphorylation in the 3 groups. For Aim 3, we will perform co-immunoprecipitation experiments using antibodies against PP2Ac followed by assessment of co-immunoprecipitated protein quantities under basal and insulin infusion in the 3 groups using the proteomics approach for protein complexes developed in our laboratory. The outcome of this research will be the discovery of abnormalities in PP2A activity, post-translational modifications, and interaction partners in skeletal muscle insulin resistance in humans, facilitating the design of new drugs to modulate PP2Ac post-translational modifications and protein-protein interactions. These drugs may restore abnormal PP2Ac activity and improve insulin signaling, resulting in the treatment and/or prevention of T2D, as well as other chronic disease.